1. Technical Field
This invention relates to the field of molecular biology. In particular, the invention relates to methods and compositions of matter for promoting stable, site-specific integration of Rep-deleted recombinant adeno-associated virus (rAAV) vectors via delivery of a functional AAV Rep gene product to the necessary location by fusing a nucleic acid encoding it to a nucleic acid encoding an intercellular trafficking “cargo” protein such as herpes simplex virus (HSV) tegument protein, VP22 or fragment thereof.
2. Description of the Background Art
Recombinant adeno-associated virus vectors have recently emerged as promising vehicles for gene transfer for a variety of reasons, including their lack of pathogenicity, wide host range, ability to transduce nonproliferating target cells, stable genomic integration, and comparatively low intrinsic immunogenicity. Genetic and sequence analyses of wild type AAV2 have demonstrated two primary open reading frames (ORFs). The left ORF is necessary for virus DNA replication, and contains two promoters at map positions 5 (p5) and 19 (p19). These promoters control expression from colinear, overlapping reading frames that arise from unspliced and spliced transcripts which produce Rep proteins of 78, 68, 52, and 40 kDa respectively. The right ORF, which is necessary for virion encapsulation, contains a single promoter at map position 40 (p40), and encodes three overlapping proteins (VP1, VP2, and VP3) with alternative translational initiation sites. The AAV coding regions are flanked by inverted terminal repeats (ITRs) which possess weak intrinsic promoter activity and are critical for DNA replication, encapsulation and host cell integration. See Berns, in “The Parvoviridae: The Viruses and Their Replication,” Fields Virology, Fields, Knipe and Howley, Eds., 3rd edition, Lippincott-Raven, 1996, pp. 2173–2197; Chatterjee and Wong, “Adeno-associated virus vectors for transduction of genes encoding ribozymes,” in Intracellular Ribozyme Applications: Principles and Protocols, Rossi and Couture (Eds.), Horizon Scientific Press, 1999; Wong and Chatterjee, “Parvovirus Vectors for Cancer Gene Therapy,” in Cancer Gene Therapy, Lattine and Gershon, Eds., Academic Press, 2000.
One of the most interesting features of wild type AAV is its ability to integrate into a specific region in human chromosome 19 termed AAVS1. Kotin et al., Proc. Natl. Acad. Sci. USA, 87:2211–2215, 1990; Samulski et al., EMBO J. 10:3941–3950, 1991. Mutational and deletion analyses have demonstrated that this property is mediated by Rep68/78, the product of the p5 promoter. Surosky et al., J. Virol. 71(10):7951–7959, 1997. Theoretically, the capacity to integrate site-specifically would be highly advantageous for rAAV vectors for several reasons. From a safety standpoint, nonrandom integration would lessen the likelihood of insertional mutagenesis. Kung et al., Curr. Top. Microbiol. Immunol. 171:1–25, 1991. In addition, cellular sequence flanking inserts are known to affect trans gene expression, resulting in varying levels of expression depending upon the location of insertion. Lacy et al., Cell 34(2):343–358, 1983. Targeted vector integration could minimize this variability of expression.
The rep gene has been removed from essentially all currently used rAAV vectors, both to provide a larger space for insertion of recombinant transgenes and to minimize the risks of recombinational events generating wild type AAV during the encapsulation process. Thus, although some studies indicate that integration is not totally random, rep-minus, wild type free rAAV stocks no longer integrate site specifically into AAVS1. Fisher-Adams et al., Blood 88:492–504, 1996; Rivadeneira et al., Int. J. Oncol. 12(4):805–810, 1998.
There is a need in the art for methods to improve the potential safety of rAAV vectors and to modify gene expression from rAAV vectors, in particular, methods which would allow site specific integration of rep-deleted rAAV vectors. Delivery of a functional AAV rep gene product to the necessary location would be of great value in achieving safer gene transfer with less unpredictable expression levels. Restoration of site-specific integration of rAAV vectors could significantly impact upon the safety and utility of rAAV vectors for gene transfer and potential gene therapy.